Assay Technique Research Articles

Hepatitis B and C viral infections and correlates among pregnant women in Ilorin, Nigeria: a cross-sectional study

Background: Hepatitis B and C are significant viral infections in pregnancy with high risk of maternal and neonatal complications. This study was conducted to determine the prevalence of hepatitis B and C viral infections and correlates among pregnant women who attended antenatal care (ANC) unit of General and Civil Service Hospitals, Ilorin, Nigeria. Methodology: This was a descriptive cross-sectional study of 282 consented pregnant women randomly selected from the ANC units of two hospitals in Ilorin. Blood samples were collected from the pregnant women and tested for hepatitis B virus (HBV) surface antigen (HBsAg) and anti-hepatitis C virus (HCV) antibody using Enzyme Linked Immunosorbent Assay (ELISA) technique. Socio-demographic and obstetrics information of the pregnant women and potential risk factors for HBV and HCV infections were collected using a pretested structured questionnaire. Statistical analysis was done using SPSS version 22.0 Results: Majority of the women were married (78.4%), from monogamous families (57.8%), do not share sharp objects (65.6%), have not received blood transfusion (65.3%), have no history of organ transplantation (91.1%), and multigravida (61.4%,) in their first (36.9%), second (37.2%) and third (25.9%) trimesters of pregnancy. Overall, 22.3% and 1.4% tested positive for HBsAg and anti-HCV respectively. Family background of the study participants showed significant (p˂0.05) association with HBsAg positivity. None of the other analysed variables showed any significant association with both HBsAg and anti-HCV positivity. Conclusion: This result showed high prevalence of HBV but low prevalence of HCV infections among pregnant women in the study setting. Improved surveillance and routine screening for HBV and HCV for women attending ANC is recommended to reduce disease burden and help achieve WHO goal of eliminating viral hepatitis as a major public health threat by 2030.

Levels of three natural milk antimicrobial proteins during the manufacture of Cheddar cheese

Variations in cheese quality are explained by many factors. Among them, some influence the microbiota of cheeses such as native milk antimicrobial proteins. This project aimed to determine the concentration and activity of lactoferrin (LF), lactoperoxidase (LP) and lysozyme (LZ) during cheesemaking. Each antimicrobial protein was analyzed by two methods. An enzyme-linked immunosorbent assay (ELISA) technique was utilized to analyze each antimicrobial protein. In addition, LP was measured by spectrometry and LZ by fluorescence. Despite the lack of correlation between the results obtained by ELISA and the other methods, the measurement of LF, LP and LZ in raw milk and thermized milk showed no significant differences. The ELISA methods were selected to determine the retention of LF, LP and LZ in fresh cheese made from thermized milk. A total of 12 independent assays of cheddar cheese made commercially by a Canadian Dairy processor and at a pilot plant were evaluated. The concentration and activity of the three antimicrobial proteins were higher in cheese than in milk by an average factor of 3.5. These results allow a better understanding of the transition of the antimicrobial proteins present in milk when making cheese and to potentially, be able to control their concentration.

Assessment <b></b>of Serum P53 Protein Level in Adult Patients with Acute Myeloid Leukemia in Correlation with Response to Treatment

Background: Acute myeloid leukemia (AML) is an adult leukemia characterized by rapid proliferation of undifferentiated myeloid precursors, leading to bone marrow (BM) failure and impaired erythropoiesis. The p53 tumor suppressor protein regulates cell division and inhibits tumor development by preventing cell proliferation of altered or damaged DNA. It orchestrates various cellular reactions, including cell cycle arrest, DNA repair, and antioxidant properties. Objectives: To investigate the relationship of P53 serum level with hematological findings, remission, and survival status in de novo AML patients. Methods: This is a cross-sectional study that enrolled 63 newly diagnosed de novo AML patients, and 15 sex- and age-matched healthy persons as a control group. Serum P53 levels were assessed using the enzyme-linked immunosorbent assay (ELISA) technique before initiating induction chemotherapy. The study was performed between November 2022 and May 2023 at the Hematology and Bone Marrow Transplant Center of the Medical City Complex in Baghdad. Results: There were significantly lower P53 serum levels in AML patients before starting chemotherapy compared to the control group. However, no substantial difference in P53 levels was identified between AML patients achieving complete remission and those exhibiting no response, nor between alive and deceased individuals. Furthermore, there was a positive yet statistically non-significant correlation between serum P53 levels and age, and no significant relationship between P53 levels and sex or various hematological parameters. Conclusion: P53 levels are low in AML patients. They are not associated with remission status or survival after six months and are not correlated with hematological values.

A critical review on phytochemicals as antiviral medications for SARS-CoV-2 virus infection.

A pandemic of acute respiratory infection, which was specified as coronavirus disease 2019, was instigated by a different strain of the virulent coronavirus SARS-CoV-2 that first appeared in late 2019. Since viral infections spread fast and there is presently no effective treatment, the use of plants with a long history of use in treating these infections has been explored regularly. The pandemic of coronavirus disease 2019 (COVID-19) has brought to light the dearth of medications with approval to treat acute viral illnesses. Because of this, the illness had a high fatality rate. The mortality rate was initially quite high and varied according to the patient's geographic location. For instance, among Chinese patients, the rate was 3·6%, whereas 1·5% of COVID-19-related deaths were documented outside of China. As of 2020, India has a 1.4% case fatality rate (CFR) of COVID-19 mortality, compared to 2.8% in Brazil and 1.8% in the USA. Many studies are being conducted to create pharmaceutical compounds specifically targeting important SARS-CoV-2 proteins. Several drug discovery initiatives are being undertaken to find powerful inhibitors by combining biochemical assay and computer-aided drug design techniques. Although plant-derived compounds have not had much success in the dominion of antivirals, plants are, however, believed to be a limitless supply of medications for a variety of diseases and clinical conditions. The scientific foundation required for developing novel natural source medications is provided by the chemical characterization and analysis of plant components. Most viral infections treated by ethnobotanical applications and historical literature on ayurveda, and traditional medicine are generally attributed to phytochemicals, which are compounds derived from medicinal plants. In this review, we have described the application of vascular plant-derived chemicals, such as tannins, polyphenols, alkaloids, and flavonoids, as antivirals, especially for managing COVID-19. This article discusses novel bioactive compounds and their molecular structures that target the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as prospective candidates for anti-coronavirus disease drugs. Moreover, to confirm the effectiveness of the phytochemicals that have demonstrated antiviral activity, clinical trials would need to be conducted in addition to the preclinical research that has already been done. To ensure spectacular findings, more applications of the compound would need to be studied to fully understand the effects of those phytochemicals whose clinical usefulness has already been established.

Anticancer activity of Lannea coromandelica on B16F10 melanoma cell line: an in vitro and molecular docking approach

BackgroundPhytochemical screening was conducted on various bark extracts of Lannea coromandelica to assess their anticancer property against the B16F10 melanoma cell line. The phytoconstituents that were previously identified were utilized in molecular docking studies against the human tyrosinase related protein 1 (TYRP1) as a target receptor in order to provide more evidence for anticancer property. MethodsBark powder was extracted by maceration method using distilled water and soxhlet extraction using ethanol. The preliminary phytochemical evaluation and determination of total phenolic and flavonoid content of both extracts were conducted using biochemical assays. The present study investigated the possible anticancer effects of ethanol and aqueous extracts on the B16F10 melanoma cell line using the MTT assay and DAPI labelling techniques. The present study employed molecular docking techniques to assess the binding interactions between phytoconstituents and the TYRP1 protein, utilizing AutoDock Vina module of PyRx 0.8 software. ResultsThe phytochemical analysis found flavonoids, steroids, terpenoids, tannins, phenolic compounds, saponins, anthraquinones, cardiac glycosides, and proteins. Ethanolic extract shown preferential cytotoxicity to B16F0 melanoma cell line in-vitro (IC50= 9.69 ± 0.68 μg/ml), while aqueous extract exhibited IC50 = 75.49 ± 5.95 μg/ml. DAPI staining showed that treated cells had altered nucleus morphology, including apoptotic bodies. According to molecular docking investigations, Quercetin has the highest binding affinity (-9.6 Kcal/mol), followed by Catechin and Myricadiol. ConclusionThe current investigation has determined that Lannea coromandelica exhibits cytotoxic characteristic, as evidenced by the utilization of computer aided drug design models and in-vitro experimentation.

Esculin Alleviates Osteoarthritis Progression Through theSirt1/NF-κB Pathway.

Osteoarthritis (OA), a joint disease associated with inflammatory processes, contributes to joint destruction. Esculin (ESC) extracted from the stem bark of Fraxinus rhynchophylla Hance has been shown to possess anti-inflammatory properties. In this study, we investigated the effect of ESC on chondrocytes treated with IL-1β and its molecular mechanism. The importance and potential mechanism of ESC in the progression of OA were evaluated. The viability of chondrocytes after exposure to ESC was examined through the CCK-8 assays. The cells were then subjected to quantitative polymerase chain reaction (qPCR), western blot, and enzyme-linked immunosorbent assay (ELISA) techniques to analyze the degradation of the extracellular matrix (ECM) and occurrence of inflammation. The NF-κB mechanism was evaluated by western blot analysis, immunofluorescence (IF), and luciferase reporter assay. Molecular docking was performed to allow for predictions on proteins that interact with ESC. Moreover, the significance of Sirt1 was explored through a knockdown experiment based on siRNA. Micro-computed tomography (CT), H&E, Safranin O-Fast Green (S-O), and immunohistochemical analyses were carried out to assess the treatment efficacy of ESC on OA in destabilization of medial meniscus (DMM) models. ESC treatment effectively inhibited ECM degradation, modulated the levels of pro-inflammatory factors, and regulated the NF-κB signaling in chondrocytes exposed to IL-1β. Mechanistically, we found that ESCs bound to Sirt1 to inhibit the activity of the NF-κB mechanism. Furthermore, ESC treatment suppressed OA progression in the DMM models. Our findings reveal that ESC ameliorates OA progression via modulating the Sirt1/NF-κB axis. This demonstrates that ESC has the potential to be applied in the treatment of OA.

Trends of coinfections among healthy blood donors: COVID-19 pandemic repercussion

ABSTRACT Background: Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) coinfection has emerged as a leading cause of morbidity throughout the world in the last two decades. The coronavirus disease 2019 (COVID-19) pandemic has escalated the disease burden further by increasing the number of intravenous (IV) drug abusers and unemployment. Aim: The present study was done to analyse the impact of COVID-19 on seroprevalence as well as trends during pre, post and pandemic years of coinfection and mono-infections in the Malwa region of Punjab. Setting and Design: This descriptive cross-sectional study was done in the department of immunohematology and blood transfusion at a tertiary care hospital. Materials and Methods: The data on transfusion-transmitted infections (TTIs) was collected for a period of four years from 2019 to 2022, that is, pre, post and during the pandemic period. All the blood samples were screened for viral markers, HIV I and II, HCV, and hepatitis B surface antigen (HBsAg) using the enzyme-linked immunosorbent assay (ELISA) technique. Malarial antigen and syphilis infection testing was done using a rapid diagnostic card test. The total number of sero-reactive cases and their distribution were noted. Results: A total of 58,953 donors were screened and included during the study period. Each blood donor was identified by a donor registration number. The overall TTI seroprevalence in blood donors was 2.83% (n = 1670). The seroprevalence of TTIs in blood donors showed an increasing trend for HIV, HCV, and HBsAg in 2019–2021, whereas there was a decrease in reactivity status in the 2022 (back to pre-pandemic year). There was a significant increase in the coinfection rate from 0.1% to 0.25%. Conclusion: The COVID-19 pandemic impact on the coinfection rate of TTI was significant. To curb these TTIs and coinfections, education and public awareness are the key factors. India is a developing country, so transfusion medicine specialists need to work day and night to practice safe blood services.

Analysis of Gliomas DNA Methylation: Assessment of Preanalytical Variables

Precision oncology is driven by biomarkers. For glioblastoma multiforme (GBM), the most common malignant adult primary brain tumor, O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is an important prognostic and treatment clinical biomarker. Time-consuming preanalytical steps such as biospecimen storage, fixation, sampling, and processing are sources of data irreproducibility, and all these preanalytical variables are confounded by intratumor heterogeneity of MGMT promoter methylation. To assess the effect of preanalytical variables on GBM DNA methylation, tissue storage/sampling (CryoGrid), sample preparation multisonicator (PIXUL), and 5-methylcytosine DNA immunoprecipitation (Matrix-MeDIP-qPCR/seq) platforms were used. MGMT promoter methylation status assayed by MeDIP-qPCR was validated with methylation-specific PCR. MGMT promoter methylation levels in frozen and formalin-fixed paraffin-embedded sample pairs were not statistically different, confirming the reliability of formalin-fixed paraffin-embedded for MGMT promoter methylation analysis. Warm ex vivo ischemia (up to 4 hours at 37 °C) and 3 cycles of repeated sample thawing and freezing did not statistically impact 5-methylcytosine at MGMT promoter, exon, and enhancer regions, indicating the resistance of DNA methylation to common variations in sample processing conditions that might be encountered in research and clinical settings. Twenty-six percent to 34% of specimens exhibited intratumor heterogeneity in the MGMT DNA promoter methylation. These data demonstrate that variations in sample fixation, ischemia duration and temperature, and DNA methylation assay technique do not have a statistically significant impact on MGMT promoter methylation assessment. However, intratumor methylation heterogeneity underscores the value of multiple biopsies at different GBM geographic tumor sites in the evaluation of MGMT promoter methylation status. Matrix-MeDIP-seq analysis revealed that MGMT promoter methylation status clustered with other differentially methylated genomic loci (eg, HOXA and lncRNAs) that are resilient to variation in the above preanalytical conditions. These observations offer new opportunities to develop more granular data-based epigenetic GBM biomarkers. In this regard, the high-throughput CryoGrid-PIXUL-Matrix toolbox could be useful.

Migration of Platinum Nanoparticles via Volatile Platinum Dioxide during Lean High-Temperature Ageing of Diesel Oxidation Catalysts

When platinum-containing diesel oxidation catalysts (DOC) are exposed to high temperatures under lean conditions, the platinum nanoparticles form volatile platinum dioxide on the catalyst surface. The exhaust flow carries the volatile platinum dioxide to the downstream aftertreatment catalyst, such as the selective catalytic reduction (SCR) catalyst, that is responsible for reducing the nitrogen oxides (NOx) emissions and can negatively impact its performance, by promoting the parasitic oxidation of ammonia. Here we investigate the factors such as exposure time, temperature and DOC design characteristics for their impact on the platinum dioxide migration, by characterising the amount of platinum deposited on the SCR catalyst at very low levels (<5 ppm), using inductively coupled plasma optical emission spectroscopy (ICP-OES) fire assay technique. Our results indicate that well-dispersed platinum, not associated with palladium, is most prone to platinum dioxide migration. We also compare several methods to suppress the platinum dioxide migration from the DOC, such as sintering of the platinum nanoparticles, stabilising the platinum nanoparticles via interaction with palladium or covering the platinum nanoparticles with a high surface area capture layer to trap the volatile platinum dioxide.

Where do we stand with screening for colorectal cancer and advanced adenoma based on serum protein biomarkers? A systematic review.

Colorectal cancer (CRC) screening has been proven to reduce both mortality and the incidence of this disease. Most CRC screening programs are based on fecal immunochemical tests (FITs), which have a low participation rate. Searching for blood protein biomarkers can lead to the development of a more accepted screening test. The aim of this systematic review was to compare the diagnostic potential of the most promising serum protein biomarkers. A systematic review based on PRISMA guidelines was conducted in the PubMed and Web of Science databases between January 2010 and December 2023. Studies assessing blood protein biomarkers for CRC screening were included. The sensitivity, specificity, and area under the ROC curve of each biomarker were collected. Among 4685 screened studies, 94 were considered for analysis. Most of them were case-control studies, leading to an overestimation of the performance of candidate biomarkers. The performance of no protein biomarker or combination of biomarkers appears to match that of the FIT. Studies with a suitable design and population, testing new assay techniques, or based on algorithms combining FIT with serum tests are needed.

Investigation of the Effect of Nano-melatonin on Oxidative Stress in Sera of Patients with Polycystic Syndrome

Background: The association between polycystic ovary syndrome (PCOS) and oxidative stress in women, the possible causes of PCOS, insulin resistance (IR), obesity, and oxidative stress. Oxidants are chemical elements that tend to gain electrons, losing positive charge. The Oxidative Stress (OS) is defined as an imbalance between the formation and synthesis of free radicals and the antioxidant defense system, which results in oxidative damage. Objective: The purpose of this research is to investigate the effect of nano-melatonin on oxidative stress in the sera of patients with polycystic syndrome. Methods: The study includes 120 Iraqi women, divided into two groups: 60 PCOS patients and 60 healthy control patients. Sample Analysis Biochemistry analyses the hormonal profile and determines the hormonal levels (LH, FSH, Testosterone, and Prolactin) that were identified for all controls and patients. Hormones analysis was performed using the enzyme-linked fluorescent assay (ELFA) technique. Synthesized ML nanoparticles by ultrasonic sonication. Results: The results indicate that the mean of serum LH significantly increased (P<0.02) in PCOS BMI ≥ 25 patients G1 (6.19±3.13) mIU/mL compared with controls C1 (4.504±1.591) mIU/mL, while a nonsignificant difference (P>0.05) when compared the BMI<25 kg/m2 for G2 (5.55±1.93) with control group C2 (6.31±2.58) of luteinizing hormone in G1 and G2. Conclusions: An increased ratio of LH to FSH was shown to be characteristic of PCOS. For PCOS, high LH values are crucial. For the purpose of diagnosing PCOS, high LH values are crucial release of gonadotropin hormones (LH, FSH) from the pituitary. To higher the production of androgen in ovarian theca cells, The LH receptor is activated by the luteinizing hormone (LH). The nano Mel was higher in the inhibition of Oxidative stress two causes nano-melatonin high surface area and small size.

Assessment of B2-Microglobulin Analysis for Renal Insufficiency in Iraqi β-Thalassemia Major Patients

Background: Beta-thalassemia is the most prevalent genetic hemoglobin apathy in the world. It is caused by a reduction or absence of beta-globin chain production, which is typically a portion of adult hemoglobin (HbA, which is α2β2). This genetic anomaly will lead to a rapid erythrocyte turnover, severe anemia, and compensatory ineffective erythropoiesis. The purpose of the current research is to reveal diagnostic, and predictive biomarkers that can be performed to detect the decline in renal function in β-thalassemia major patients with early stage renal impairment with high sensitivity and specificity, ascertain changes in B2-microglobulin as a biomarker in β-thalassemia patient. Objective: The samples were collected from Karama Hospital-Genetic Hematology Center/Baghdad during the period from the 1st of September 2022 until the 1st of January 2023. Methods: The patient group consisted of 45 patients with β-thalassemia major with repeated blood transfusion and the control group consisted of 45 individuals who seemed to be healthy. Age ranged between (18-35) years (for patients and control). The serum samples were used to measure biochemical parameters, S.Creatinine, blood urea, and Urine B2-microglobulin were measured from urine samples. B2-microglobulin level in Urine was estimated by Sandwich enzyme-linked immunosorbent assay (ELISA) technique, while serum Blood Urea and Serum Creatinine concentration were calculated by Colorimetric Kit by Spectrophotometer method, and Ferritin level in blood was estimated by BioMrieux Mini Vidas. Results: The statistical examination was carried out using SPSS software. B2-microglobulin biomarkers in the thalassemia Patient group were significantly higher than those in the control group (p<0.001). While serum ferritin, B.Urea and S.Cr increased in patient groups compared to the control group (p<0. 005). Conclusions: All parameters included in this study are significantly higher in β-thalassemia patients than in healthy subjects. Renal hemosiderosis and asymptomatic renal dysfunction are prevalent among β-thalassemia major patients with repeated blood transfusions, which are not found in routine renal investigations.

BAFF as a Potential Marker for Toxoplasma Gondii Seroprevalence Among Cancer Patients on Chemotherapy

Background: Toxoplasmosis is a global zoonotic disease, that is mainly asymptomatic in healthy persons, but critical and life-threatening in immunocompromised patients. The infection is caused by an opportunistic obligate intracellular parasite protozoon “Toxoplasma gondii” (T. gondii) that can cause fatal complications in immunosuppressed patients. Cancer patients are being immunocompromised from the cancer itself and on the other hand cancer therapy they receive may reactive a latent T. gondii infection that leaves them at high risk. The B-cell activating Factor (BAFF) is a cytokine that plays an essential role in the activation, maturation, and survival of B cells. It is vital for the appropriate functioning of the immune system. Monitoring BAFF levels in cancer patients undergoing chemotherapy could be important for preventing or controlling infections in these vulnerable patients. Objectives: This study aimed to detect seroprevalence of T. gondii in cancer patients receiving chemotherapy and assess the levels of BAFF in those patients. Materials and Methods: 150 serum samples were collected from oncology patients having different types of cancer and receiving chemotherapy, from different ages. And 30 samples from healthy people for control. In this cross-sectional study, samples were collected from the 1st of October 2023 to the 1st of March 2024, at Kirkuk Oncology and Hematology center. The method used was the Sandwich Enzyme-Linked Immunosorbent Assay (Sandwich-ELISA) technique. Results: the overall anti-T. gondii IgG and IgM seropositive samples were found to be 67 (37.2%) in the total study population and 67(44.7%) in cancer patients. BAFF levels were elevated in cancer patients infected with T. gondii compared with the cancer patients with no infection and the healthy control group. A significant difference was indicated with (p-value 0.0004). Conclusions: A high rate of T. gondii seroprevalence was revealed among oncology patients receiving chemotherapy with elevated levels of BAFF associated with T. gondii infection in those patients.

Molecular and Immunological Detection of Hepatitis C Virus in Patients with Chronic Renal Failure

Due to its association with hepatocellular carcinoma and being one of the ten most common malignancies worldwide, hepatitis C viral infection has become a severe public health concern. Therefore, establishing an accurate, reliable and sensitive diagnostic test for this infection is strongly advised. Real-time polymerase chain reaction (PCR) has been created to achieve this purpose. The current study was established to investigate the hepatitis C virus among Iraqi patients with chronic renal failure and to detect the virus immunologically by the fourth generation enzyme-linked immunosorbent assay technique and molecularly by real-time PCR. As a result, out of 50 patients with chronic renal failure undergoing dialysis, 39 patients tested positive for anti-HCV IgG by ELISA test and only 9 patients of the total count tested positive and showed growth curves with copy numbers of 1×102, 2.5×102, 3×102, 2.7×103, 2.7×103, 3.5×103, 8.7×104, 3.5×105 and 5.5×106 copies/ml respectively detected for HCV only by real-time PCR. Therefore, real-time PCR is beneficial for precise viral load determination and detection of viral hepatitis in chronic renal failure patients.

Lymph node metastasis diagnosis of postoperative OSCC patients by analyzing extracellular vesicles in drainage fluid based on microfluidic isolation

Lymph node metastasis (LNM) is a typical marker in oral squamous cell carcinoma (OSCC) indicating poor prognosis. Pathological examination by artificial image acquisition and analysis, as the main diagnostic method for LNM, often takes a week or longer which may cause great anxiety of the patient and also retard timely treatment. However, there are few efficient fast LNM diagnosis methods in clinical applications currently. Our previous study profiled the proteomics of extracellular vesicles (EVs) derived from postoperative drainage fluid (PDF) and showed the potential of detecting specific EVs that expressed aspartate β-hydroxylase (ASPH) for LNM diagnosis in OSCC patients. Considering that the analysis of ASPH+ PDF-EVs is challenging due to their low abundance (counting less than 10% of total EVs in PDF) and the complex EV isolation process of ultra-centrifugation, we developed a facile platform containing two microfluidic chips filled with antibody-modified microbeads to isolate ASPH+ PDF-EVs, with both the capture and retrieval rate reaching around 90%. Clinical sample analysis based on our method revealed that a mean of 6 × 106 /mL ASPH+ PDF-EVs could be isolated from LNM+ OSCC patients compared to 2.5 × 106 /mL in LNM- OSCC ones. When combined with enzyme-linked immunosorbent assay (ELISA) technique that was commonly used in clinical laboratories in hospitals, this microfluidic platform could precisely distinguish postoperative OSCC patients with LNM or not in several hours, which were validated by a double-blind test containing 6 OSCC patients. We believe this strategy has promise for early diagnosis of LNM in postoperative OSCC patients and finally helps guiding timely and reasonable treatment in clinic.

Multienzymatic Orthogonal Activation of DNA Codec Enables Tumor-Specific Imaging of Base Excision Repair Activity.

Accurate monitoring of base excision repair (BER) activity in cancer cells is critical for advancing the comprehension of DNA repair processes, gaining insights into cancer development, and guiding treatment strategies. However, current assay techniques for assessing BER activity in cancer cells face challenges due to the heterogeneous origins and diversity of BER enzymes. In this work, we present a highly reliable triple loop-interlocked DNA codec (GATED) that enables precise assessment of BER activity in cancer cells through signal amplification mediated by multienzyme orthogonal activation. The GATED device features a dumbbell-shaped DNA probe to encode two BER enzymes for BER-related signal conversion as well as two bound circular DNA to decode the apurinic/apyrimidinic sites for apurinic/apyrimidinic endonuclease 1 (APE1)-mediated signal amplification. Importantly, GATED is orthogonally activated by multiple target BER enzymes (i.e., uracil DNA glycosylase, thymine DNA glycosylase, and APE1), resulting in a unified fluorescent signal that significantly improves the detection specificity and sensitivity to BER enzymes. Additionally, we demonstrate that the GATED has exceptional biostability within complex biological systems, where it was successfully employed to monitor BER activity in cancer cells with high specificity and enabled cell-based high-throughput screening for BER inhibitors. The GATED provides a much-needed tool for the real-time monitoring of BER activity and the screening of BER inhibitors in cancer cells, potentially advancing both the investigation and clinical application of BER biology.

A noninvasive method of diagnosing membranous nephropathy using exosomes derived from urine.

Membranous nephropathy (MN) is a specific autoimmune disease affecting kidneys. It is characterized by the accumulation of immune complexes in the glomerular basement membrane. Renal biopsy is currently the standard procedure to confirm the diagnosis, although the presence of autoantibodies against the phospholipase A2 receptor (PLA2R) can also help diagnose. In this study, we aimed to investigate the potential of urinary exosomes as noninvasive markers for diagnosing MN. Exosomes were extracted from urine samples of five patients with MN and four healthy controls. The concentration of PLA2R was measured in both urine and isolated exosomes using enzyme-linked immunosorbent assay techniques. The measurements were adjusted based on the urine creatinine (UCr) level of each participant. The levels of PLA2R/UCr were investigated in urine and urine-derived exosomes from patients and controls. Results of the analysis revealed significantly higher expression of PLA2R/UCr in patients compared to the control group (p < 0.05). Furthermore, the expression level of PLA2R/UCr was higher in urine-derived exosomes than in urine samples. Additionally, a positive correlation was observed between the expression levels of PLA2R/UCr and the urine protein-to-creatinine ratio, with urine-derived exosomes exhibiting a stronger correlation than urine samples. Studies have indicated that measuring exosomal PLA2R/UCr levels in urine could be a noninvasive method for diagnosing MN. Using urine-derived exosomes could also reduce the burden of performing a biopsy on patients and facilitate follow-up treatment, such as monitoring for future recurrence.

Investigating the immunological activity of the Hsp70-P113 fusion protein for Mycoplasma ovipneumoniae detection: a groundbreaking study

BackgroundMycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi.MethodsThis study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni–NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized.ResultsThe resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%.ConclusionThe results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.

Proteomic profiling of oleamide-mediated polarization in a primary human monocyte-derived tumor-associated macrophages (TAMs) model: a functional analysis.

Tumor-associated macrophages (TAMs) play a critical function in the development of tumors and are associated with protumor M2 phenotypes. Shifting TAMs towards antitumor M1 phenotypes holds promise for tumor immunotherapy. Oleamide, a primary fatty acid amide, has emerged as a potent anticancer and immunomodulatory compound. However, the regulatory effects of oleamide on TAM phenotypes remain unclear. We used real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to study the influence of oleamide on primary human monocyte-derived TAM phenotypes, and we investigated the protein expression profiles based on mass spectrometry to analyze the effect of oleamide on macrophage polarization. Moreover, the advantageous binding scores between oleamide and these target candidate proteins are examined using molecular docking. Our study revealed that oleamide effectively suppressed the M2-like TAM phenotype by reducing interleukin (IL)-10 production and downregulating M2-like markers, including vascular endothelial growth factor A (VEGFA), MYC proto-oncogene, bHLH transcription factor (c-Myc), and mannose receptor C-type 1 (CD206). Moreover, the conditioned medium derived from oleamide-treated TAMs induces apoptosis of MDA-MB-231 breast cancer cells. Proteomic analysis identified 20 candidate up- and down-regulation proteins targeted by oleamide, showing modulation activity associated with the promotion of the M1-like phenotype. Furthermore, molecular docking demonstrated favorable binding scores between oleamide and these candidate proteins. Collectively, our findings suggest that oleamide exerts a potent antitumor effect by promoting the antitumor M1-like TAM phenotype. These novel insights provide valuable resources for further investigations into oleamide and macrophage polarization which inhibit the progression of breast cancer, which may provide insight into immunotherapeutic approaches for cancer.

Hydro-Ethanol Extracts of Xylopia aethiopica Enhanced Serum Testosterone Levels in Male Adult Wistar Rats

Objectives To investigate the effects of hydro-ethanolic extracts of leaves and fruit with or without seeds on male sex hormones. Material and Methods Hydro-ethanolic extracts of the leaves, fruits with seeds, and fruits without seeds were processed using the Soxhlet extraction technique. Wistar rats were administered 125, 250, and 500 mg/kg of the extracts by oral gavage for 30, 60, and 90 days while the control group received feeds and water ad libitum. After the treatment periods, rats were sacrificed by cervical dislocation, and blood was collected by cardiac puncture. Serum follicle-stimulating hormones (FSHs), luteinising hormone (LH), progesterone, prolactin, and testosterone levels were determined using an enzyme-linked immunosorbent assay technique. Data were compared using analysis of variance (ANOVA), and a p-value of 0.05 was considered significant. Results Serum testosterone, FSH, and LH levels in rats treated with 125, 250, and 500 mg/kg were significantly increased (p &lt; 0.05) in a dose-dependent manner compared with controls. Conclusion The consumption of this commonly used food spice appears to have beneficial effects on the male sex hormones.